Bioactive peptides, uses thereof and process for the production of same

ABSTRACT

Novel bioactive peptide compositions and process for producing the same and the use of such compositions for enhancing the growth of warm blooded animals and fish is disclosed.

FIELD OF THE INVENTION

[0001] This invention relates to novel bioactive peptide compositionswhich enhance the growth of animals, a process for the preparation ofsame and the use of such compositions for enhancing the growth ofwarmblooded animals and fish.

BACKGROUND OF THE INVENTION

[0002] It is well known that porcine and bovine plasma protein can beused in feed formulations for warm blooded animals to improve health,growth and general performance. However, the need for a replacement forsuch plasma protein is of great importance. The cost of plasma proteincontinues to increase as foreign and domestic demand increases. Inaddition, inadequate supply of plasma protein is a frequent problem.Also, due to concern of contamination of host animals through thefeeding of plasma protein there is becoming a further need for anon-plasma product which can be utilized in those formulations whereplasma protein is either not available or its use barred.

[0003] In addition, it is well known that protein hydrolyzates preparedby reaction of selected protein materials with at least one proteolyticenzyme can also be used in feed formulations. Such compounds aregenerally in such amounts as to maximize its content in the feedformulation without having a negative effect on the host. Suchhydrolyzates provided a replacement for fish meal as a protein sourceand thus while providing nutritional values provided no other benefit inthe growth of the host.

[0004] Thus there is presently experienced a need for a suitablereplacement for plasma as an ingredient of feed formulations as well asthe need to achieve growth enhancement for host animals.

SUMMARY OF THE INVENTION

[0005] The aforementioned need is met by the present invention byproviding a bioactive peptide composition which not only provides analternative to plasma products but also serves to enhance the growth ofboth animals and fish.

[0006] According to the present invention a bioactive peptidecomposition is produced by the enzymatic hydrolysis of a protein usingthe proteolytic enzyme derived from cod, specifically the stomachs ofAtlantic cod.

[0007] The present invention is based upon the discovery that thepeptide product obtained through enzymatic hydrolysis of a proteinsource with the pepsin enzyme derived from the stomach of Atlantic codcan be used as a substitute for plasma protein in achieving growthenhancement of warm blooded animals.

[0008] In a first aspect of the present invention there is providednovel animal feed compositions which enhance growth.

[0009] In addition, the present invention is also based on the furtherdiscovery that such peptide products can be used at low levels toenhance the growth of fish.

[0010] Accordingly, in one aspect of the present invention there isprovided a novel peptide product which is useful for enhancing thegrowth of warm blooded animals and fish.

[0011] In another aspect of this invention there is provided a processfor the production of growth enhancing peptides from a protein source.

[0012] In another aspect of this invention there is provided a novelprocess for enhancing the growth of warm blooded animals and fish.

[0013] Other aspects, objects and the several advantages of thisinvention will be apparent from the foregoing disclosure and appendedclaims.

[0014] The term bioactive peptide compositions as used herein isintended to define a composition consisting essentially of a mixture ofpeptides with aromatic amino acids in N-terminal position, produced byhydrolysis of a protein source at a pH of 2-6 with pepsins from fish asthe hydrolytic enzyme.

[0015] The bioactive peptide product of this invention is prepared by aprocess comprising the steps of:

[0016] (a) hydrolyzing a selected protein material, dispersed in anaqueous medium, together with cod pepsin at a controlled temperature andacidic pH;

[0017] (b) removing lipids and solids from the pepsin treated acidizedprotein source; and thereafter

[0018] (c) recovering the resulting bioactive peptide.

[0019] The process may employ protein material from any of a greatvariety of sources including meats, fish and plants. Protein materialfrom fish is particularly preferred for use in the process of thisinvention.

[0020] The process of this invention is particularly adapted toeffecting protein hydrolysis in an acidic medium of controlled pH toproduce a bioactive peptide composition suitable for use in theenhancement of growth of host animals. Protein material is prepared fortreatment by grinding, comminution, or other means to provide a suitableparticle size for slurring with acid. The aqueous slurry is heated withagitation at a combination of temperature and time such that the initialreaction velocity of hydrolysis is maximized.

[0021] The enzyme employed in the process of this invention is theproteolytic pepsin enzyme derived from the stomach of Atlantic cod. Inone presently preferred embodiment of this invention the enzyme isobtained by a process which comprises homogenizing the cod stomach inthe presence of enough formic acid to lower the pH to 4 and then heatingfor a period of time whereby the homogenate becomes a liquid slurry dueto the digestion of the tissues by the enzymes present in the stomachand thereafter recovering from the aqueous phase following removal ofsludge and oils following concentration and drying the desired pepsinenzyme.

[0022] In carrying out the process, the homogenate of the Atlantic codstomach can also be employed as the enzyme source. Thus following thehomogenizing of the cod stomach in the presence of acid and thedigestion of the tissues thereof by the enzymes present therein, theresulting liquid slurry which is enzyme rich can be employed directlyinto the process of the present invention as the enzyme source for theproduction of bioactive peptides.

[0023] In the production of the bioactive peptide composition of theinvention, a suitable protein source is treated at a pH in the range ofabout 2-6, preferably at a pH of 4, with the pepsin enzyme derived fromthe Atlantic cod stomachs for a time to effect hydrolysis of the proteinsource so as to form a mixture of peptides having aromatic amino acidsin N-terminal positions which following heating and thereafter removalof oils and sludge provided a liquid composition consisting essentiallyof bioactive peptides and deactivated cod pepsin.

[0024] The hydrolysis step is conducted in a continuous stirred-tankreactor employing an enzyme to protein substrate ratio selected toobtain the desired level of conversion within a time period of 24 to 100hours.

[0025] Generally the enzyme is added in amounts in the range of 0.5 to 5g per 1000 kg of protein.

[0026] Acid employed in the hydrolysis process in an amount to providenormally 3% volume by volume.

[0027] Following the removal of oils and undissolved solids from thepartially hydrolyzed aqueous protein material dispersion, the recoveredprotein hydrolyzate fraction may be concentrated, as by reverse osmosisor thermal evaporation and dried, as by freeze drying or spray drying,and sent to product storage for subsequent packaging and distribution.

[0028] Pepsin enzyme suitable for use in one embodiment of the presentinvention is obtained by hydrolysis of Atlantic cod fish stomachs at apH in the range of 2-6. Following hydrolysis, the resulting hydrolyzateis subjected to lipid and solids removal followed by ultrafiltration ofthe resulting liquid peptide pepsin mixture to effect recovery of thepepsin enzyme therefrom.

[0029] The Atlantic cod pepsin which is used in the process of thisinvention is a proteolytic enzyme prepared from Atlantic cod (Gadusmorhua) stomach by autolysis. The enzyme has a specific activity of100-200 Anson Units expressed as μ mol TCA-soluble tyrosine releasedfrom haemoglobin per hour at pH 3 and 25° C.

[0030] The compositions produced by the process of this invention havebeen found to possess significantly distinctive properties. Suchcompositions are bioactive peptides which are capable of enhancinggrowth when included in feed compositions for host animals.

[0031] Accordingly, the bioactive peptides of this invention are mostappropriate for providing a process for the enhancement of growth of ananimal by feeding such animal with an amount of such bioactive peptideto effect growth enhancement of the host animal. In general such growthenhancement is achieved when the bioactive peptide is introduced intothe host animal in an amount in the range of 0.1 to 5 g/kg body weightper day.

[0032] Feed formulations using the active peptide compositions of thisinvention will be added to conventional feed ingredients for theintended host animal an amount of bioactive peptide which is sufficientto achieve growth enhancement activity.

[0033] Generally such amounts will be in the range of from about 0.1 toabout 5 weight percent of the total feed, preferably from about 1 to 2%.

[0034] Enzyme hydrolysis of the protein source is normally carried outat a temperature in the range of about 10° C. to about 45° C. and at apH in the range of about 2 to about 6. Suitable pH is achieved by use ofeither a mineral or organic acid or salts thereof which is added priorto addition of the pepsin. The amount of acid employed is that whichwill provide the desired pH level.

[0035] In one presently preferred embodiment of the invention there isemployed a lower carboxylic acid namely formic acid.

[0036] Protein source starting materials suitable for the process of thepresent invention are:

[0037] 1. fish wastes including whole trash fish, fish left afterfilleting, fish solubles, fish viscera and any other materials which areby-products of the fishing industry and processing

[0038] 2. pork skins and tissues

[0039] 3. beef tissue

[0040] 4. soy bean proteins or other seed proteins

[0041] 5. milk proteins

[0042] The following examples will further illustrate the process andproducts of the invention.

EXAMPLE I Enzyme Preparation

[0043] Fish stomachs of Atlantic cod, collected from processing plants,are homogenized in the presence of enough formic acid to lower the pH ofthe homogenate to 4. The homogenate is then heated to 27° C. and held atthis temperature for 48 hours. During this period the homogenate becomesa liquid slurry due to digestion of the tissues by the enzymes presentin the stomachs. The liquid slurry is then stored in a tank wherein asludge gradually deposits at the bottom and oil floats to the surface.The aqueous phase between the sludge and the oil layer is thenconcentrated by ultrafiltration (cut off 10,000 mw) and sprayed dried ata temperature below the inactivation temperature for the pepsin enzyme(65° C. The resulting dried and yellow powder contains 100-200 Ansonunits of protease activity as measured by standard assays for pepsinactivity e.g. μ mol tyrosine released from haemoglobin per hour at pH 3and 25° C.

EXAMPLE II Production of Liquid Bioactive Peptides

[0044] 1000 Kg of by-product from fish processing, e.g. residual meat onback bones, skins, guts, heads, were homogenized in an industrial meatgrinder and 30 liters of 85% formic acid added when the homogenate waspumped into a storage tank with stirring equipment.

[0045] To the resulting acidized homogenate was added 1 kg of codstomach enzymes which was obtained in accordance with the protocol ofExample I. The resulting mixture was then maintained at 30° C. for aperiod of about 5 days to achieve enzymatic degradation of the acidizedby products.

[0046] Thereafter, the resulting digest was heated to a temperature of80° C. to inactivate the added enzyme and to permit oil which waspresent in the raw materials to float to the surface for removal. Afterremoval of oil by centrifugation in an industrial centrifuge and removalof undissolved bones by screening there was obtained 850-950 liters ofan aqueous slurry which was then concentrated by evaporation to 40% dryweight and a final volume of 250-350 liters.

[0047] The resulting concentrated product containing 20% by weight ofnon digested protein and 70 weight percent of peptide and 10% mineralsis designated liquid crude peptide product or <<LCP>>.

EXAMPLE III Production of Dried Bioactive Peptide

[0048] 100 liters of the liquid crude peptide product of Example II wasdried by spray drying in a NIRO spray dryer at an air out temperature of80° C., to provide 40 kg of dried crude peptide product <<DCP>>containing undigested protein, peptides and amino acids.

EXAMPLE IV Production of Refined Peptides <<RBP>>

[0049] 400 liters of LCP produced as described in Example II wassubjected to conventional particle filtration using a bag filtrationunit, to provide about 250 liters of a solution free of particles biggerthan 40μ. Thereafter the resulting filtrate was subjected toultrafiltration in an industrial hollow fiber cartridge (Amicon)filtration unit with a cut off of 10,000 MW to provide 200 liters of apermeate containing peptides with MW lower than 10,000. The resultingRefined Bioactive Peptide <<RBP>> can be used in the liquid form.However, 200 liters of RBP was dried by spray drying in a Niro dryer at85° C. (air out) to provide 80 kg of RBP in dry form.

EXAMPLE V Preparation of Feed Compositions

[0050] Using dried refined peptide product as produced in Example IV, aseries of test feed compositions were prepared using a standard dietformulation having the peptide content as noted in the following table.For performance evaluation a further series of feed compositions wasprepared in the same manner with porcine plasma protein. Composition No.1 2 3 4 5 6 7 Feeding Period 0-13 days Plasma protein % 0 4 3 2 1 0 0Refined Peptide % 0 0 1 2 3 4 2 Feeding Period 13-33 days Plasma protein% 0 0 0 0 0 0 0 Refined Peptide % 0 0 0 0 0 2 1

EXAMPLE VI The Effect of RBP and Porcine Plasma Protein

[0051] To determine the effect of peptide (RBP)and porcine plasma ongrowth performance of starter pigs a series of treatments using theformulations of Example V were carried out with 7 pens of 22 individualstarter pigs.

[0052] The results of the feeding treatments are shown in the followingtable: Treatment composition 1 2 3 4 5 6 7 Weight (kg) Initial 5.91 5.905.91 5.91 5.91 5.91 5.91 Day 13 7.58 8.18 8.53 8.29 9.17 8.94 8.23 Day33 16.24 16.24 17.04 16.85 17.94 16.49 17.8 Weight Gain (kg) 0-13 days1.67 2.28 2.62 2.37 3.26 3.03 2.32 0-33 days 10.33 10.34 11.13 10.9412.03 10.58 11.89 Weight Gain (%) 0-13 days 23 38 44 40 55 51 39 0-33days 175 175 188 185 204 179 201 Protein 0.32 0.27 0.29 0.26 0.26 0.27consumed/ weight gain

[0053] The above results show that use of the bioactive peptide of thepresent invention has a marked effect on growth of young pigs and thatthis product can replace plasma proteins in starter diets. At a level of2% in the starter diets the bioactive peptide (RBP) causes the sameenhancement of growth as 4% porcine plasma protein during the first 13days after weaning.

[0054] Unlike plasma protein, the bioactive peptide of the presentinvention results in enhanced growth throughout the entire growth periodfrom weaning of the pigs and the following 33 days.

EXAMPLE VII Preparation of Fish Feed Compositions

[0055] A composition for use as a feed for salmon was prepared byadmixing the following ingredients: Ingredient % of dry weight Fish Meal(low temperature dried) 44 Fish meal (normal quality) 19 Fish oil 20Wheat meal 14 Potato starch 2 Vitamin C 0.1 Premix (minerals andvitamins) 1.0 Carrophyll pink (astaxanthin) 0.1 Chemical composition Drymatter 96% Protein 52% Lipid 21% Minerals 10%

[0056] For demonstration of the effectiveness of bioactive peptide inthe salmon feed, a composition was prepared wherein 5% of the fish mealwas replaced by 5% of dried bioactive crude peptide (DCP ).

EXAMPLE VIII Effect of DCP on Salmon Growth

[0057] Salmon (Salmo salar) at an average individual weight of 600 gramswere separated in two net cages in the sea with 100 fish in each cage.One group was fed the standard salmon feed of Example VII and the othergroup the same feed containing 5% DCP.

[0058] After feeding for 6 months the salmon fed the standard salmonfeed had an average weight of 2.280 kg whereas the salmon fed the 5% DCPdiet had an average weight of 2.630 kg. The growth in the referencegroup during this feeding period was in other words from an averageindividual weight of 600 grams to 2,280 g, or 1,680 grams whereas theDCP containing group grew from 600 grams to 2,630 grams, or 2,030 grams.

[0059] Comparison of the growth of the two groups indicates that usingthe product of the present invention there was achieved a 12% growthover those fishes that did not receive the bioactive peptide (DCP).

We claim:
 1. A process for production of bioactive peptide compositionswhich comprises: a. treating a protein source with an acid; b.contacting the resulting acid treated protein source with pepsin enzymederived from fish; c. removing lipids from the pepsin treated acidizedprotein source; d. removing solids from the pepsin treated source andthereafter; e. recovering the resulting bioactive peptide compositions.2. The process of claim 1 wherein said acid is a mineral or organic acidor salts thereof.
 3. The process of claim 2 wherein said mineral acid isselected from the group consisting of hydrochloric, phosphoric andsulphuric acids.
 4. The process of claim 2 wherein said acid is anorganic acid selected from the group consisting of formic, acetic,propionic and citric acids.
 5. The process of claim 1 wherein saidprotein source is fish.
 6. The process of claim 1 wherein said pepsinenzyme is derived from the stomach of Atlantic cod.
 7. The process ofclaim 1 wherein said contacting with pepsin enzyme is carried out underconditions suitable to effect the formation of bioactive peptides havingaromatic amino acids in N-terminal position.
 8. The process of claim 1wherein step (a) is carried out at a pH in the range of 2-6, atemperature in the range of 10° C. to 60° C. and for a time sufficientto effect peptide formation.
 9. The bioactive peptide product of theprocess of claim 1 .
 10. A process for the production of growthenhancing peptides which comprises enzymatically hydrolyzing a proteinsource with pepsin source derived from fish at a pH in the range of fromabout 2 to
 6. 11. The process of claim 10 wherein the enzyme hydrolysisof said protein source is carried out under conditions suitable for theproduction of a mixture of peptides having aromatic amino acids inN-terminal position.
 12. The process of claim 11 wherein said aromaticamino acids is at least one of the groups consisting of tyrosine, phenylalanine and arginine.
 13. The process of claim 10 wherein said pepsinenzyme is derived from the stomach of Atlantic cod.
 14. The process ofclaim 13 wherein said enzyme hydrolysis is carried out for a period inthe range of 24 to 100 hours.
 15. The process of claim 14 wherein saidhydrolyzing is carried out at a temperature in the range of from 10° C.to 60° C.
 16. The process of claim 10 wherein the enzymatic producedpeptides are recovered as a product of the process.
 17. A bioactivepeptide composition consisting essentially of a mixture of peptideshaving an aromatic amino acid in the N-terminal position, produced byenzymatic hydrolysis of a protein source at pH in the range of 1-6 withpepsin derived from fish as the hydrolytic enzyme.
 18. The compositionof claim 17 wherein said peptide consists of less that about 100 aminoacid units and has a molecular weight below 10,000.
 19. The compositionof claim 17 wherein said aromatic amino acid is at least one acidselected form the group consisting of tyrosine, phenylalanine andarginine.
 20. The composition of claim 17 wherein said protein source isfish.
 21. The composition of claim 17 wherein said hydrolytic enzyme isderived from the stomach of Atlantic cod.
 22. A process for theenhancement of growth of an animal which comprises feeding said animalwith an amount sufficient to effect growth of a bioactive peptidecomposition consisting essentially of a mixture of peptides having anaromatic amino acid in the N-terminal position, produced by enzymatichydrolysis of a protein source at a pH in the range of 2-6 with pepsinfrom fish as the hydrolytic enzyme.
 23. The process of claim 22 whereinsaid animal is at least one of the group consisting of warm bloodedanimals and fish.
 24. The process of claim 23 wherein said animal is apig.
 25. The process of claim 23 wherein said animal is a fish.
 26. Afeed composition for animals which will enhance the growth thereof, saidcomposition containing therein a bioactive peptide compositionconsisting essentially of a mixture of peptides having an aromatic aminoacid in the N-terminal position, produced by enzymatic hydrolysis of aprotein source at a pH in the range of 2-6 with pepsin from fish as thehydrolytic enzyme.
 27. The feed composition of claim 26 wherein saidbioactive peptide composition is present in an amount in the range offrom 0.1 to 5 weight percent.
 28. The feed composition of claim 26wherein said animals are one of the group consisting of warm bloodedanimals or fish.
 29. The feed composition of claim 28 wherein said warmblooded animal is a pig.
 30. The feed composition of claim 26 whereinsaid animal is a fish
 31. Use of the bioactive peptide derived from aprotein source by the enzymatic hydrolysis thereof with the pepsinenzyme derived for Atlantic cod for the enhancement of growth of a warmblooded animal or fish.
 32. The use in accordance with claim 31 whereinthe animal or fish is fed from 0.1 to 5 grams of bioactive peptide perkg of body weight.